A program of research is proposed that will provide labeled small molecules: amino acids, folates, and antifolates for the study by NMR spectroscopy of the interactions of these compounds with enzymes that utilize folate or its reduced forms as cofactors. The label of interest for proton magnetic resonance spectroscopy (PMR) is deuterium (D) in place of hydrogen (H), and for carbon magnetic resonance spectroscopy (CMR) is 13C in place of 12C. The labels placed in appropriate locations in amino acids are incorporated into enzymes of L. casei by fermentation. The labeled folates and antifolates are studied with and without labeled dihydrofolate reductase and thymidylate synthetase to reveal details of the mechanisms of action of these two enzymes. These results should be useful in the design of new antifolates as potential cancer chemotherapeutic agents.